Plant cell wall degradation by saprophytic Bacillus subtilis strains: gene clusters responsible for rhamnogalacturonan depolymerization

Abstract

Plant cell wall degradation is a premier event when Bacillus subtilis, a typical saprophytic bacterium, invades plants. Here we show the degradation system of rhamnogalacturonan type I (RG-I), a component of pectin from the plant cell wall, in B. subtilis strain 168. Strain 168 cells showed a significant growth on plant cell wall polysaccharides such as pectin, polygalacturonan, and RG-I as a carbon source. DNA microarray analysis indicated that three gene clusters (yesOPQRSTUVWXYZ, ytePQRST, and ybcMOPST-ybdABDE) are inducibly expressed in strain 168 cells grown on RG-I. Cells of an industrially important bacterium, B. subtilis strain natto, fermenting soybeans also express the gene cluster including the yes series during the assimilation of soybean used as a carbon source. Among proteins encoded in the yes cluster, YesW and YesX were found to be novel types of RG lyases releasing disaccharide from RG-I. Genetic and enzymatic properties of YesW and YesX suggest that strain 168 cells secrete YesW, which catalyzes the initial cleavage of the RG-I main chain, and the resultant oligosaccharides are converted to disaccharides through the extracellular exotype YesX reaction. The disaccharide is finally degraded into its constituent monosaccharides through the reaction of intracellular unsaturated galacturonyl hydrolases YesR and YteR. This enzymatic route for RG-I degradation in strain 168 differs significantly from that in plant-pathogenic fungus Aspergillus aculeatus. This is, to our knowledge, the first report on the bacterial system for complete RG-I main chain degradation.

FIG. 1.

Assimilation of pectin-related polysaccharides by B. subtilis. (A) Structure of pectin. Symbols of sugars are indicated in the box. (B) Growth of strain 168 cells on different carbon sources: glucose (closed circles), pectin (open triangles), polygalacturonan (closed triangles), and RG-I (closed squares). The nonaddition of saccharides as a carbon source was used as the negative control (open circle). Bacterial growth was determined by measuring turbidity (optical density at 600 nm [OD₆₀₀]).

FIG. 2.

B. subtilis strain natto. (A) The soybean surface was covered with strain natto cells. The LB plate surface was streaked with natto and incubated at 37°C. (B) Growth of strain natto cells on boiled soybean minimal medium. Viable cells in culture broth were determined by measuring colony counts after spreading on LB medium. (C) Genetic organization of the yes cluster. (D) Expression of 16S rRNA, YesW, and YesX genes in strain natto cells grown on soybeans. Each gene was amplified by using total RNAs after treatment with (+RT) and without (−RT) reverse transcriptase.

FIG. 3.

Properties of RG lyases. (A) SDS-PAGE of purified YesW and YesX followed by protein staining with Coomassie brilliant blue. Lane M contained molecular mass standards (from top to bottom)—synthetic polypeptides with molecular masses of 250, 150, 100, 75, 50, 37, and 25 kDa. Lane 1, purified recombinant YesW (5 μg protein); lane 2, purified recombinant YesX (5 μg protein). The arrow indicates the position of each enzyme. (B) Enzymatic degradation of RG-I. Degradation of the RG chain by YesW (upper) and YesX (lower). The reaction mixture consisted of 5 mg/ml of the RG chain, 50 mM of Tris-HCl (pH 7.5), 2 mM of CaCl₂, and the purified enzyme. Reaction products were periodically sampled and analyzed on TLC plates, followed by staining with sulfuric acid. Reaction times (min) are indicated in the figure. The spots indicated by “2s” are unsaturated RG disaccharides with and without an addition of ammonium ion, which was used during preparation of the RG chain. Galacturonic acid (GalA) and rhamnose (Rha) were used for marker control. (C) Effects of pH and temperature on the activity and stability of YesW and YesX. The relative activity of YesW is indicated by a closed circle and solid line and that of YesX by an open circle and broken line. For optimal pH (left panel), activity was assayed with HEPES-NaOH (pH 6.5 and 7.2), Tris-HCl (pH 7.5, 8.0, and 8.5), and glycine-NaOH (pH 9.0, 10.0, and 11.0). Activity at pH 8.0 in YesW and pH 8.5 in YesX is taken as 100%. For optimal temperature (center panel), activity at 65°C in YesW and 60°C in YesX is taken as 100%. For thermal stability (right panel), purified enzymes were preincubated for 5 min at the temperatures indicated and residual enzyme activity was measured. The activity of enzymes preincubated at 4°C for 5 min is taken as 100%.

FIG. 4.

Degradation profiles of RG chain with RG lyases. (A) Analysis of final products from the RG chain through the RhgB, YesW, or YesX reaction by size-exclusion chromatography. Each product was analyzed after a 60-min reaction by Superdex peptide 10/300 GL. Product from the RG chain without enzyme added was used as the negative control. All profiles are overlaid, and the smallest product through the YesW or YesX reaction is indicated by an arrow. Degradation profiles of the RG chain with YesW (B) and YesX (C) were periodically analyzed. Reaction times were 0, 5, 20, and 60 min. The smallest product is indicated by an arrow. (D) ESI-MS spectrum of the smallest product released from the RG chain through the YesX reaction. The main peak in the negative mode coincided with the molecular mass of the deprotonated ion from the unsaturated RG disaccharide indicated in the box.

FIG. 5.

Bacterial and fungus systems for degradation of the RG main chain. (A) B. subtilis. (B) A. aculeatus. Thin arrows indicate the cleavage site for RG-I-degrading enzymes, and thick arrows indicate the degradation pathway. Details are given in the text.